Project Summary/Abstract Recent evidence has highlighted the critical importance of the perivascular niche in the bone marrow as the main site where hematopoietic stem and progenitor cells (HSPCs) reside. Studies have increasingly shown that an alteration in the bone marrow (BM) niche components can promote blood disease initiation and progression. Our research focus is on a key component of the vascular niche: bone marrow endothelial cells (BMECs). Previous studies have shown the importance of BMECs in promoting HSPC engraftment and reconstitution. Our goal is to understand how an abnormality in the niche can impact angiogenesis, hematopoiesis and blood disorders. To this end, a novel inducible mouse model was generated in our laboratory to specifically introduce a KRas or NF1 mutation in endothelial cells. Preliminary data indicate that this mutation significantly impacted hematopoiesis in a manner reminiscent of myeloproliferative disorders. To test our hypothesis, we will inject adult mice with Tamoxifen to activate KRasG12D signaling in BMECs. To complement this study, we will also transplant BM cells from wild-type (WT) mice into lethally irradiated KRasG12D and WT mice to define the effects of non-hematopoietic endothelial KRasG12D signaling on hematopoiesis and angiogenesis. Complete blood counts, flow cytometry and histopathology techniques will be used to inform us of any hematopoietic change. To investigate how an abnormal endothelial niche impacts the functional output of hematopoiesis, in Aim 1, we will perform a competitive transplant with BM cells from KRasG12D mice or controls along with competitor cells into lethally irradiated WT recipients. Our preliminary data suggest that KRasG12D mice have altered chimerism in all blood lineages. Our transplant studies, complemented with in vitro studies will provide a better understanding on how a niche alteration can modify the hematopoietic landscape, thereby biasing hematopoiesis toward the myeloid lineage. In Aim 2, molecular studies will shed light on which cellular pathways and signaling components are involved. Our initial studies suggest that an abnormal endothelial niche promotes the secretion of pro-angiogenic cytokines from HSPCs, which also have decreased quiescence. As research has shown that patients with blood disorders have abnormal vasculature in their BM, confocal microscopy and related software will be used to examine any abnormal vasculature and microvessel density. It is crucial to understand how each niche component can be a contributing factor. As limited scholarship exists on the contribution of abnormal BMECs in blood diseases, our research is intended to bridge that gap in knowledge. Only by considering both aberrant blood cells and their niche would it be possible to achieve more effective therapy in patients that are resistant to chemotherapy. Our research thus has broad implications in the field of blood disorders. Clinically, this could help to identify new targets and to develop better therapeutic strategies in order to improve survival and prognosis in myeloproliferative disorders/myelodysplastic syndromes and other blood disease patients.